Name: retroviral scanning: mapping mlv integration sites to define cell-specific regulatory regions
Uploaded: 2017-05-28T14:00:00
Description: Watch this Scientific Journal Video about Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions at JoVE.com

Dette arbejde blev støttet af tilskud fra Det Europæiske Forskningsråd (ERC-2010-AdG, GT-SKIN), det italienske ministerium for uddannelse, universiteter og forskning (FIRB-Futuro i Ricerca 2010-RBFR10OS4G, FIRB-Futuro i Ricerca 2012-RBFR126B8I_003 , EPIGEN Epigenomics Flagskibsprojekt), Det Italienske Sundhedsministerium (Unge forskere Ring 2011 GR-2011-02352026) og Imagine Institute Foundation (Paris, Frankrig).

1. MLV-transduktion af humane celler <ol><li> Isoler målceller og transducer dem med en mLV-afledt retroviral vektor, der huser eGFP-reportergenet og pseudotypes med vesikulært stomatitisvirus G (VSV-G) eller det amfotrope kuvertglykoprotein 16 . <ol><li> Hold mock-transducerede celler som en negativ kontrol for de følgende analyser. Da mLV-baserede retrovirale vektorer kan transducere effektivt delende celler, dyrker målcellepopulationen i tilstande, der stimulerer cellede...

<ol>
	<li>Ernst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+analysis+of+chromatin+state+dynamics+in+nine+human+cell+types.">Mapping and analysis of chromatin state dynamics in nine human cell types.</a> Nature. 473 (7345), 43-49 (2011).</li><li>Shlyueva, D., Stampfel, G., Stark, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+enhancers:+from+properties+to+genome-wide+predictions.">Transcriptional enhancers: from properties to genome-wide predictions.</a> Nat Rev Genet. 15 (4), 272-286 (2014).</li><li>Roadmap Epigenomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Heintzman, N. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancers+are+major+targets+for+murine+leukemia+virus+vector+integration.">Enhancers are major targets for murine leukemia virus vector integration.</a> J Virol. 88 (8), 4504-4513 (2014).</li><li>LaFave, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mLV+integration+site+selection+is+driven+by+strong+enhancers+and+active+promoters.">mLV integration site selection is driven by strong enhancers and active promoters.</a> Nucleic Acids Res. 42 (7), 4257-4269 (2014).</li><li>Gupta, S. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+genomic+environment+of+the+provirus+determines+the+abundance+of+HTLV-1-infected+T-cell+clones.">The host genomic environment of the provirus determines the abundance of HTLV-1-infected T-cell clones.</a> Blood. 117 (11), 3113-3122 (2011).</li></ol>

Her beskrev vi en protokol for genomgående kortlægning af integrationsstederne for mLV, et retrovirus, der målretter chromatinområder, epigenetisk markeret som aktive promotorer og enhancere. Kritiske trin og / eller begrænsninger af protokollen omfatter: (i) mLV-transduktion af målcellepopulationen; (Ii) amplifikation af virus-værtsforbindelser med LM-PCR; (Iii) hentning af en høj del af integrationssteder. MLV-baserede retrovirale vektorer transducerer effektivt dividende celler. Den lave effektivitet ved tran...

Celleidentitet bestemmes ved ekspression af specifikke gener af gener. Rollen af ​​cis-regulatoriske elementer, såsom promotorer og forstærkere, er afgørende for aktiveringen af ​​specifikke transkriptionsprogrammer fra celletypen. Disse regulatoriske regioner er kendetegnet ved specifikke chromatin-egenskaber, såsom særlige histon-modifikationer, transkriptionsfaktorer og co-faktorer bindende og chromatin tilgængelighed, som i vid udstrækning er blevet anvendt til deres genom-bred identifikation i flere celletyper 1 , 2 , 3 . Især benyttes den genomgående profil af acetylering af histon H3-lysin 27 (H3K27ac) almindeligt til at definere aktive promotorer, forstærkere og superforstærkere 4 , 5 , 6 . Moloney-muse-leukæmivirus (MLV) er et gamma-retrovirus, der er meget anvendt til genOverførsel i pattedyrsceller. Efter inficering af en målcelle omdannes det retrovirale RNA-genom retro-transkriberet i et dobbeltstrenget DNA-molekyle, som binder virale og cellulære proteiner til at samle præintegrationskomplekset (PIC). PIC kommer ind i kernen og binder værtscellechromatinet. Her medierer den virale integrase, en nøgle-PIC-komponent integrationen af ​​det provirale DNA i værtscellegenomet. MlV-integration i det genomiske DNA er ikke tilfældigt, men forekommer i aktive cis-regulatoriske elementer, såsom promotorer og enhancere, på en cellespecifik måde 7 , 8 , 9 , 10 . Denne særlige integrationsprofil er medieret af en direkte interaktion mellem mLV integrasen og det cellulære bromodomain og extraterminalt domæne (BET) proteinerne 11 , 12 , 13 . BET proteiner (BRD2, BRD3 ogBRD4) virker som en bro mellem værtschromatin og mLV PIC: gennem deres bromodominer genkendes de stærkt acetylerede cis-regulatoriske regioner, mens extraterminalt domænet interagerer med mLV integrase 11 , 12 , 13 . Her beskriver vi retroviral scanning, et nyt værktøj til kortlægning af aktive cis-regulatoriske regioner baseret på integrationen af ​​mLV. Kort fortalt transduceres celler med mLV-afledt retroviral vektor, der udtrykker det forstærkede grønne fluorescerende protein (eGFP) reportergen. Efter genomisk DNA-ekstraktion amplificeres krydsningerne mellem den 3&#39;-lange terminale gentagelse (LTR) af mlV-vektoren og det genomiske DNA ved hjælp af linkermedieret PCR (LM-PCR) og massivt sekventeret. MlV-integrationssteder kortlægges til det humane genom og genomiske regioner, der er højt målrettet af mLV, defineres som klynger af mLV-integrationssteder. Retroviral scanningNing blev anvendt til at definere celle-specifikke aktive regulatoriske elementer i adskillige humane primære celler 14 , 15 . MLV-klynger co-mapped med epigenetisk definerede promotorer og enhancers, hvoraf de fleste indeholdt aktive histonmærker, såsom H3K27ac, og var celle-specifikke. Retroviral scanning tillader genomfattende identifikation af DNA-regulatoriske elementer i prospektivt rensede cellepopulationer 7 , 14 såvel som i efterfølgende definerede cellepopulationer, såsom keratinocytstamceller, der mangler effektive markører til fremtidig isolering 15 .

<table>
	<tbody>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>ThermoScientific</td>
			<td>10010031</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoScientific</td>
			<td>16000044</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>0.2 ml tubes</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tubes</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAGEN QIAmp DNA mini Kit&#160;</td>
			<td>QIAGEN</td>
			<td>51306</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>T4 DNA ligase&#160;</td>
			<td>New England BioLabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Reaction buffer</td>
			<td>New England BioLabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr>
			<td>Linker Plus Strand oligonucleotide</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-PO4-TAGTCCCTTAAGCGGAG-3&#8217;&#160; (Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>Linker Minus Strand oligonucleotide</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GTAATACGACTCACTATAGGGCTCCGCTTAAGGGAC-3&#8217; (Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>Tru9I</td>
			<td>Roche-Sigma-Aldrich</td>
			<td>11464825001</td>
			<td></td>
		</tr>
		<tr>
			<td>SuRE/Cut Buffer M</td>
			<td>Roche-Sigma-Aldrich</td>
			<td>11417983001</td>
			<td></td>
		</tr>
		<tr>
			<td>PstI&#160;</td>
			<td>Roche-Sigma-Aldrich</td>
			<td>10798991001</td>
			<td></td>
		</tr>
		<tr>
			<td>SuRE/Cut Buffer H</td>
			<td>Roche-Sigma-Aldrich</td>
			<td>11417991001</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Taq DNA Polimerase High Fidelity&#160;</td>
			<td>Invitrogen</td>
			<td>11304011</td>
			<td></td>
		</tr>
		<tr>
			<td>10mM dNTP Mix</td>
			<td>Invitrogen</td>
			<td>18427013</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well thermal cycler (with heated lid)</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>linker primer</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GTAATACGACTCACTATAGGGC-3&#8217; (Purification grade: PCR grade)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR primer</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GACTTGTGGTCTCGCTGTTCCTTGG-3&#8217; (Purification grade: PCR grade)</td>
		</tr>
		<tr>
			<td>linker nested primer 454</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GCCTTGCCAGCCCGCTCAG[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR nested primer 454</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#8217;-GCCTCCCTCGCGCCATCAGTAGC[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>linker nested primer Illumina</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#39;-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[AGGGCTCCGCTTAAGGGAC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>MLV-3&#8217; LTR nested primer Illumina</td>
			<td>general lab supplier</td>
			<td></td>
			<td>5&#39;-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[GGTCTCCTCTGAGTGATTGACTACC](Purification grade: SDS-PAGE)</td>
		</tr>
		<tr>
			<td>Sodium Acetate Solution (3M) pH 5.2</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (absolute) for molecular biology</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Topo TA Cloning kit (with pCR2.1-TOPO vector)</td>
			<td>Invitrogen</td>
			<td>K4500-01</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction kit</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Ethidium bromide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>Invitrogen</td>
			<td>15628019</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>6X Loading Buffer</td>
			<td>ThermoScientific</td>
			<td>R0611</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td>ThermoScientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT Index kit</td>
			<td>Illumina</td>
			<td>FC-131-1001 or FC-131-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>2x KAPA HiFi Hot Start Ready Mix&#160;</td>
			<td>KAPA Biosystems</td>
			<td>KK2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynal magnetic stand for 2 ml tubes</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP 60 ml kit</td>
			<td>Beckman Coulter Genomics</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl 10 mM, pH 8.5</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2200 TapeStation system</td>
			<td>Agilent Technologies</td>
			<td>G2964AA</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5582</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>D1000 Reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5583</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kit for Illumina platforms (ABI Prism)</td>
			<td>KAPA Biosystems</td>
			<td>KK4835</td>
			<td></td>
		</tr>
		<tr>
			<td>ABI Prism 7900HT Fast Real-Time PCR System</td>
			<td>Applied Biosystems</td>
			<td>4329003</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH 1.0 N, molecular biology-grade</td>
			<td>general lab supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HT1 (Hybridization Buffer)</td>
			<td>Illumina&#160;</td>
			<td>Provided in the MiSeq Reagent Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v3 (150 cycles)</td>
			<td>Illumina</td>
			<td>MS-102-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>MiSeq System</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td></td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>FC-110-3001</td>
			<td></td>
		</tr>
	</tbody>
</table>

retroviral scanning: mapping mlv integration sites to define cell-specific regulatory regions

Moloney-murine leukæmi (MLV) virusbaserede retrovirale vektorer integrerer overvejende i acetylerede forstærkere og promotorer. Af denne grund kan mLV integration sites bruges som funktionelle markører af aktive regulerende elementer. Her præsenterer vi et retroviralt scanningsværktøj, som muliggør genomspændende identifikation af cellespecifikke forstærkere og promotorer. Kort fortalt transduceres målcellepopulationen med en mlV-afledt vektor, og genomisk DNA spaltes med et hyppigt skærende restriktionsenzym. Efter ligering af genomiske fragmenter med en kompatibel DNA-linker tillader linker-medieret polymerasekædereaktion (LM-PCR) amplifikationen af ​​virus-værtsgenomforbindelserne. Massiv sekventering af ampliconerne anvendes til at definere mLV-integrationsprofilgenomet bredt. Endelig defineres klynger af tilbagevendende integrationer for at identificere cellespecifikke regulatoriske regioner, der er ansvarlige for aktiveringen af ​​specifikke transkriptionsprogrammer af celletype. <p class= &quot;Jove_content&quot;&gt; Det retrovirale scanningsværktøj tillader genomfattende identifikation af cellespecifikke promotorer og forstærkere i fremtidigt isolerede målcellepopulationer. Navnlig repræsenterer retroviral scanning en instrumentteknik til retrospektiv identifikation af sjældne populationer ( fx somatiske stamceller), der mangler robuste markører til fremtidig isolering.

Arbejdsstrøm af den retrovirale scanningsprocedure Arbejdsstrømmen for retroviral scanningsproceduren er skematiseret i figur 1 . Målcellepopulationen renses og transduceres med en mLV-afledt retroviral vektor, der udtrykker et eGFP-reportergen. Transgenet flankeres af de to identiske lange terminale gentagelser (5&#39;- og 3&#39;-LTR), der sikrer syntese, revers transkription og integration af vi...

Watch this Scientific Journal Video about Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions at JoVE.com

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Her beskriver vi en protokol for genomgående kortlægning af integrationen af ​​Moloney-murine leukæmivirusbaserede retrovirale vektorer i humane celler.

Retroviral Scanning: Kortlægning af MLV Integration Sites til at definere Cell-specifikke Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

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